I. Field of the Invention
The present invention relates to the fields of molecular and cell biology generally, and more specifically, it addresses mechanisms for growth control in eurkaryotic cells. In particular, there are provided genes that inhibit normal cell death and methods for use thereof.
II. Related Art
The control of host cell translation, and often the control of replication, are integral parts of the life cycle of a virus. However, recent evidence suggests that most eurkaryotic cells respond to viral disruption of normal cellular physiology by undergoing programmed cell death (apoptosis) (White, 1993). To counteract this, many viruses have evolved mechanisms to block host cell death (Clem and Miller, 1994a; White and Gooding, 1994). In several cases, viral genomes have been found to contain genes whose products interact with proteins that play a central role in regulating cell survival.
For example, the product of the crmA gene isolated from cowpox virus can inhibit apoptosis through its ability to inhibit members of the interleukin-1xcex2-converting enzyme (ICE) protease family (Miura et al., 1993; Yuan et al., 1993; Gagliardini et al., 1994; Komiyama et al., 1994; Kumar et al., 1994; Wang et al., 1994; Fernandes-Alnemri et al., 1995; Los et al., 1995; Tewari et al., 1995). The E1B 19 kD protein of adenovirus also can protect cells from death induced by a wide variety of stimuli (Debbas and White, 1993; Sabbatini et al., 1995). The ability of E1B 19 kD to protect cells from apoptosis correlates with its ability to bind to members of the bcl-2 family that promote cell death (Chiou et al., 1994; Farrow et al., 1995).
Insect baculoviruses contain at least two independent genes that promote the survival of the infected host cell-p35 (Friesen and Miller, 1987; Clem et al., 1991; Hershberger et al., 1992; Clem and Miller, 1993; Kamita et al., 1993; Hershberger et al., 1994) and iap (Crook et al., 1993; Birnbaum et al., 1994). Recombinant baculoviruses lacking both genes induce accelerated host cell death leading to severely impaired virus production (Clem and Miller, 1994b). Although p35 and iap have no sequence similarity, they are functionally equivalent in the context of the virus. When provided in trans either gene can protect host cells from death induced by a baculovirus lacking both genes (Clem and Miller, 1994b). This suggests that p35 and iap exert their effects at key points in the cellular apoptotic pathway.
Consistent with this view is the discovery that p35 can confer protection from cell death in mammalian cells (Rabizadeh et al., 1993; Beidler et al., 1995), an observation which reflects the high degree of evolutionary conservation of the apoptotic cell death pathway (Vaux et al., 1994; White et al., 1994). Recently, p35 has been shown to act by blocking the activity of members of the ICE family of cysteine proteases (Bump et al., 1995; Xue and Horvitz, 1995). Whether iap also blocks an evolutionarily conserved step in apoptosis has not been determined. The iap genes isolated from different baculoviruses all display similar structural features.
The utility of proteins that are capable of inhibiting apoptosis are manifold. First, such proteins, or their corresponding genes, may be used to immortalize cell lines that otherwise would perish during culture. This makes possible not only the study of these cells, but also presents the option of growing these cells in large numbers in order to isolate protein species therefrom. Second, the identification of iap""s and their function permits the possible intervention, in a clinical setting, when these proteins are interfering with normal programmed cell death, or apoptosis. This may be accomplished by providing an ilp inhibitor or an antisense nucleic acid that interferes with the expression of an ilp protein. Thus, the identification of novel proteins having these activities and uses provide important new tools for those working in this arena.
It is, therefore, an object of the present invention to provide new ilp proteins and gene coding therefor. It also is an object of the present invention to provide methods of using these new proteins, for example, in the immortalization of cells for culture, for inhibiting the activation of cysteine proteases and to sustain host cell survival following viral infection. It also is contemplated that, through the use of other technologies such as antisense expression and antibody treatment, one can treat certain cancers by inhibiting the effects of these new ilp proteins.
Therefore, there is provided an isolated and purified ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has at least one of the activities of (i) inhibition of apoptosis, (ii) inhibition of cysteine protease activation and (iii) inhibition of virus-induced cell death. In a preferred embodiment, the ilp will have all three of these activities. In another embodiment, the ring finger domain is carboxy-terminal to the BIR domains, and in yet another embodiment, an amphipathic domain separates the BIR domains and the ring finger domain. The amphipathic domain preferably is between about 120 and 170 amino acids. The ilp may be a human protein, as in FIG. 2C (SEQ ID NO:2). The ilp may be a Drosophila protein, as in FIG. 2B (SEQ ID NO:1).
In another embodiment, there is provided an isolated and purified polynucleotide encoding an ilp, the ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has at least one of the activities of (i) inhibition of apoptosis, (ii) inhibition of cysteine protease activation and (iii) inhibition of virus-induced cell death. The polynucleotide may be of human or Drosophila origin.
In yet another embodiment, a recombinant host cell comprising a polynucleotide encoding an ilp, said ilp comprising at least three BIR domains and a ring finger domain, wherein said ilp has at least one of the activities of (i) inhibition of apoptosis, (ii) inhibition of cysteine protease activation and (iii) inhibition of virus-induced cell death.
In still yet another embodiment, there is provided an antibody that is immunologically reactive with an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has at least one of the activities of (i) inhibition of apoptosis, (ii) inhibition of cysteine protease activation and (iii) inhibition of virus-induced cell death.
In still yet another embodiment, there is provided a method for producing an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has at least one of the activities of (i) inhibition of apoptosis, (ii) inhibition of cysteine protease activation and (iii) inhibition of virus-induced cell death, comprising the steps of (a) providing a recombinant host cell comprising a polynucleotide encoding said ilp and (b) culturing said recombinant host cell. In a further step, there is provided a step of isolating the ilp.
In still yet another embodiment, there is provided a method for inhibiting apoptosis comprising the step of providing to a cell an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has the activity of inhibition of apoptosis. The method preferably comprises contacting by transforming said cell with said polynucleotide. Transforming may be accomplished by viral-mediated gene transfer, receptor-mediated gene transfer, liposome-mediated gene transfer, calcium phosphate-mediated gene transfer or direct gene injection.
In still yet another embodiment, there is provided a method for inducing apoptosis comprising blocking the expression in a cell of an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has the activity of inhibition of apoptosis. The method preferably comprises providing to a cell an antisense polynucleotide corresponding to a portion of the ilp. The providing preferably comprises transforming the cell, and the transforming may be by viral-mediated gene transfer, receptor-mediated gene transfer, liposome-mediated gene transfer, calcium phosphate-mediated gene transfer and direct gene injection.
In still yet another embodiment, there is provided a method for inhibiting apoptosis comprising increasing the level in a cell of an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has the activity of inhibition of apoptosis. In one method, the increasing comprises providing to the cell an ilp. Alternatively, the increasing comprises stimulating the expression of an endogenous ilp. In this embodiment, the cell may be transformed with a polynucleotide encoding the ilp. The transforming may be by viral-mediated gene transfer, receptor-mediated gene transfer, liposome-mediated gene transfer, calcium phosphate-mediated gene transfer and direct gene injection.
In still yet another embodiment, there is provided a method for inhibiting cysteine protease activation comprising the step of providing to a cell an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has the activity of inhibition of cysteine protease activation. Alternatively, the method may comprise the step of providing to a cell a polynucleotide encoding an ilp, the ilp comprising at least three BIR domains and ring finger domain, wherein the ilp has the activity of inhibition of cysteine protease activation.
In still yet another embodiment, there is provided a method for inhibiting virus-induced cell death comprising the step of providing to a cell an ilp comprising at least three BIR domains and a ring finger domain, wherein the ilp has the activity of inhibition of virus-induced cell death. Alternatively, the method may comprise the step of providing to a cell a polynucleotide encoding an ilp, the ilp comprising at least three BIR domains and ring finger domain, wherein the ilp has the activity of inhibition of virus-induced cell death.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.